Skip to main content

Advertisement

SpringerLink
Log in

Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana

  • Biotechnologically Relevant Enymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Aguilar CF, Sanderson I, Moracci M, Ciaramella M, Nucci R, Rossi M, Pearl LH (1997) Crystal structure of the β-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability. J Mol Biol 271:789–802

    Article  CAS  Google Scholar 

  • Bauer MW, Kelly RM (1998) The family 1 β-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism. Biochemistry 37:17170–17178

    Article  CAS  Google Scholar 

  • Bauer MW, Bylina EJ, Swanson RV, Kelly RM (1996) Comparison of a β-glucosidase and a β-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 271:23749–23755

    Article  CAS  Google Scholar 

  • Belancic A, Gunata Z, Vallier MJ, Agosin E (2003) β-Glucosidase from the grape native yeast Debaryomyces vanrijiae: purification, characterization, and its effect on monoterpene content of a Muscat grape juice. J Agric Food Chem 51:1453–1459

    Article  CAS  Google Scholar 

  • Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Bronnenmeier K, Kern A, Liebl W, Staudenbauer WL (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl Environ Microbiol 61:1399–1407

    Article  CAS  Google Scholar 

  • Chhabra SR, Shockley KR, Conners SB, Scott KL, Wolfinger RD, Kelly RM (2003) Carbohydrate-induced differential gene expression patterns in the hyperthermophilic bacterium Thermotoga maritima. J Biol Chem 278:7540–7552

    Article  CAS  Google Scholar 

  • Dong W, Yinbo QU, Peiji G (1996) Transglycosylation of extracellular β-glucosidase of Trichoderma pseudokoningii S38 and its function in regulation of cellulase biosynthesis. J Gen Appl Microbiol 42:363–369

    Article  Google Scholar 

  • Ducret A, Trani M, Lortie R (2002) Screening of various glycosidases for the synthesis of octyl glucoside. Biotechnol Bioeng 77:752–757

    Article  CAS  Google Scholar 

  • Duffaud GD, McCutchen CM, Leduc P, Parker KN, Kelly RM (1997) Purification and characterization of extremely thermostable β-mannanase, β-mannosidase, and α-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068. Appl Environ Microbiol 63:169–177

    Article  CAS  Google Scholar 

  • Esen A (1993) β-Glucosidases: overview, in β-glucosidases. In: Esen A (ed) Biochemistry and molecular biology, 1st edn. American Chemical Society, Washington, DC, pp 1–14

    Google Scholar 

  • Fourage L, Dion M, Colas B (2001) Kinetic study of a thermostable β-glycosidase of Thermus thermophilus: effects of temperature and glucose on hydrolysis and transglycosylation reactions. Glycoconj J 17:377–383

    Article  Google Scholar 

  • Gabelsberger J, Liebl W, Schleifer KH (1993) Purification and properties of a recombinant β-glucosidase of the hyperthermophilic bacterium Thermotoga maritima. Appl Microbiol Biotechnol 40:44–52

    Article  CAS  Google Scholar 

  • Gusakov AV, Sinitsyn AP, Klesov AA, Goldshteins GK (1984) Reactions of hydrolysis and transglycosylation catalyzed by cellobiase: kinetics and mathematical model of the process. Biokhimiya 49:1110–1120

    CAS  Google Scholar 

  • Kaper T, Lebbink JH, Pouwels J, Kopp J, Schulz GE, van der Oost J, de Vos WM (2000) Comparative structural analysis and substrate specificity engineering of the hyperthermostable β-glucosidase CelB from Pyrococcus furiosus. Biochemistry 39:4963–4970

    Article  CAS  Google Scholar 

  • King MR, Yernool DA, Eveleigh DE, Chassy BM (1998) Thermostable α-galactosidase from Thermotoga neapolitana: cloning, sequencing and expression. FEMS Microbiol Lett 163:37–42

    CAS  PubMed  Google Scholar 

  • Liebl W, Gabelsberger J, Schleifer KH (1994) Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases. Mol Gen Genet 242:111–115

    Article  CAS  Google Scholar 

  • Lodge JA, Maier T, Liebl W, Hoffmann V, Strater N (2003) Crystal structure of Thermotoga maritima α-glucosidase AglA defines a new clan of NAD+-dependent glycosidases. J Biol Chem 278:19151–19158

    Article  CAS  Google Scholar 

  • Moracci M, Capalbo L, Ciaramella M, Rossi M (1996) Identification of two glutamic acid residues essential for catalysis in the β-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus. Protein Eng 9:1191–1195

    Article  CAS  Google Scholar 

  • Nucci R, Moracci M, Vaccaro C, Vespa N, Rossi M (1993) Exo-glucosidase activity and substrate specificity of the β-glycosidase isolated from the extreme thermophile Sulfolobus solfataricus. Biotechnol Appl Biochem 17:239–250

    CAS  PubMed  Google Scholar 

  • Painbeni E, Valles S, Poliana J, Flors A (1992) Purification and characterization of a Bacillus polymyxa β-glucosidase expressed in Escherichia coli. J Bacteriol 174:3087–3091

    Article  CAS  Google Scholar 

  • Perez-Pons JA, Rebordosa X, Querol E (1995) Properties of a novel glucose-enhanced β-glucosidase purified from Streptomyces sp. (ATCC11238). Biochim Biophys Acta 1251:145–153

    Article  Google Scholar 

  • Petzelbauer I, Reiter A, Splechtna B, Kosma P, Nidetzky B (2000) Transgalactosylation by thermostable β-glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus. Binding interactions of nucleophiles with the galactosylated enzyme intermediate make major contributions to the formation of new beta-glycosides during lactose conversion. Eur J Biochem 267:5055–5066

    Article  CAS  Google Scholar 

  • Raasch C, Streit W, Schanzer J, Bibel M, Gosslar U, Liebl W (2000) Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase. Extremophiles 4:189–200

    Article  CAS  Google Scholar 

  • Rojas A, Arola L, Romeu A (1995) β-Glucosidase families revealed by computer analysis of protein sequences. Biochem Mol Biol Int 35:1223–1231

    CAS  PubMed  Google Scholar 

  • Sanz-Aparicio J, Hermoso JA, Martinez-Ripoll M, Lequerica JL, Poliana J (1998) Crystal structure of β-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases. J Mol Biol 275:491–502

    Article  CAS  Google Scholar 

  • Sunna A, Moracci M, Rossi M, Antranikian G (1997) Glycosyl hydrolases from hyperthermophiles. Extremophiles 1:2–13

    Article  CAS  Google Scholar 

  • Takashima S, Nakamura A, Hidaka M, Masaki H, Uozumi T (1999) Molecular cloning and expression of the novel fungal β-glucosidase genes from Humicola grisea and Trichoderma reesei. J Biochem 125:728–736

    Article  CAS  Google Scholar 

  • Trincone A, Perugino G, Rossi M, Moracci M (2000) A novel thermophilic glycosynthase that effects branching glycosylation. Bioorg Med Chem Lett 10:365–368

    Article  CAS  Google Scholar 

  • Voorhorst WGB, Eggen RLL, Lensink EJ, Vos WMDE (1995) Characterization of celB gene encoding for β-glucosidase from the hyperthermophilic archeon Pyrococcus furiosus and its expression and site directed mutation in Escherichia coli. J Bacteriol 177:7105–7111

    Article  CAS  Google Scholar 

  • Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W (1995) Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431–444

    Article  CAS  Google Scholar 

  • Yan TR, Liau JC (1998) Synthesis of cello-oligosaccharides from cellobiose with β-glucosidase II from Aspergillus niger. Biotechnol Lett 20:591–594

    Article  CAS  Google Scholar 

  • Yernool DA, McCarthy JK, Eveleigh DE, Bok JD (2000) Cloning and characterization of the glucooligosaccharide catabolic pathway β-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana. J Bacteriol 182:5172–5179

    Article  CAS  Google Scholar 

  • Zechel DL, Boraston AB, Gloster T, Boraston CM, Macdonald JM, Tilbrook DM, Stick RV, Davies GJ (2003) Iminosugar glycosidase inhibitors: structural and thermodynamic dissection of the binding of isofagomine and 1-deoxynojirimycin to beta-glucosidases. J Am Chem Soc 125:14313–14323

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by Korea Research Foundation (2003-042-F20015). The help of Prof. Kim, Myo-Jung at Inje University, with HPAEC measurements is gratefully acknowledged. T.-H. Park is the recipient of a graduate student fellowship supported by the Brain Busan 21 project.

Author information

Authors and Affiliations

  1. Department of Microbiology, College of Natural Sciences, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, South Korea

    Tak-Hyun Park, Ki-Won Choi, Ho-Young Kang & Jaeho Cha

  2. Department of Food Science and Biotechnology & Institute of Life Sciences and Resources, Kyunghee University, Yongin, 449-701, South Korea

    Cheon-Seok Park

  3. Department of Food Science and Nutrition, Yonsei University, Seoul, 120-749, South Korea

    Soo-Bok Lee

  4. Department of Chemistry and Center for Innovative Bio-Physio Sensor Technology, Pusan National University, Busan, 609-735, South Korea

    Kwang-Jae Shon & Jang-Su Park

Authors
  1. Tak-Hyun Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ki-Won Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cheon-Seok Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Soo-Bok Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ho-Young Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kwang-Jae Shon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jang-Su Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jaeho Cha
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jaeho Cha.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, TH., Choi, KW., Park, CS. et al. Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana . Appl Microbiol Biotechnol 69, 411–422 (2005). https://doi.org/10.1007/s00253-005-0055-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-005-0055-1

Keywords

Search

Navigation